Conformational Flexibility of p150Glued(1-191) Subunit of Dynactin Assembled with Microtubules.

ABSTRACT

Microtubule (MT)-associated proteins perform diverse functions in cells. These functions are dependent on their interactions with MTs. Dynactin, a cofactor of dynein motor, assists the binding of dynein to various organelles and is crucial to the long-distance processivity of dynein-based complexes. The largest subunit of dynactin, the p150Glued, contains an N-terminus segment that is responsible for the MT-binding interactions and long-range processivity of dynactin. We employed solution and magic angle spinning NMR spectroscopy to characterize the structure and dynamics of the p150Glued N-terminal region, both free and in complex with polymerized MTs. This 191-residue region encompasses the cytoskeleton-associated protein glycine-rich domain, the basic domain, and serine/proline-rich (SP-rich) domain. We demonstrate that the basic and SP-rich domains are intrinsically disordered in solution and significantly enhance the binding affinity to MTs as these regions contain the second MT-binding site on the p150Glued subunit. The majority of the basic and SP-rich domains are predicted to be random coil, whereas the segments S111-I116, A124-R132, and K144-T146 in the basic domain contain short α-helical or ß-sheet structures. These three segments possibly encompass the MT-binding site. Surprisingly, the protein retains a high degree of flexibility upon binding to MTs except for the regions that are directly involved in the binding interactions with MTs. This conformational flexibility may be essential for the biological functions of the p150Glued subunit.